It's so often the case — processes that were previously considered separate and distinct turn out to be intimately related. Whereas post-transcriptional silencing occurs through RNA interference (RNAi), transcriptional silencing is associated with 'silent' heterochromatin, which is mediated by histone methylation. The RNA-induced initiation of transcriptional gene silencing (RITS) complex is known to be required for heterochromatic gene silencing, but it also contains short interfering (si)RNAs that are generated by Dicer (Dcr), an enzyme that is involved in RNAi. In Nature Genetics, Noma et al. report the discovery of a self-enforcing loop mechanism through which RITS binding to heterochromatic loci enables the RNAi machinery to function in cis to destroy aberrant RNAs and generate siRNAs for heterochromatin maintenance.

Noma et al. began by showing, using chromatin immunoprecipitation (ChIP) analysis and immunofluorescence, that all three known subunits of the RITS complex — Argonaute, Chp1 and Tas3 — stably associate with all known heterochromatic domains in the Schizosaccharomyces pombe genome, such as centromeres, telomeres and the mat locus.

RITS is known to be essential for heterochromatin assembly at centromeres, so the authors investigated its possible role in heterochromatin assembly at the mat locus. They found that RITS cooperated with Atf1 and Pcr1, which belong to the ATF-CREB (cAMP-responsive-element-binding protein) family, to nucleate heterochromatin assembly at the mat locus. Mapping of RITS at the mat locus showed that, surprisingly, RITS could spread from the initial nucleation site, a centromere-homologous (cenH) repeat, to the neighbouring sequences. This presumably allows RITS to exert control over sequences that are incapable of initiating an RNAi response. For RITS to spread from cenH throughout the mat locus, though, Swi6 — a protein that mediates spreading of Lys9-methylated histone H3 — was required. Dcr-generated siRNAs, though, were not required for this spreading, but were needed for the initial targeting of RITS to cenH. The authors found that cenH was transcribed bidirectionally, and that the level of cenH transcript was regulated by both transcriptional and post-transcriptional silencing mechanisms: a lack of Swi6 (which is dispensable for RITS targeting at cenH but is required for transcriptional silencing) or a lack of Dcr (which isn't needed for heterochromatin maintenance at the mat region but does process dsRNAs into siRNAs) caused cenH transcripts to accumulate — substantially so in double mutants.

Noma et al. also investigated whether the stable association of RITS with heterochromatic loci was essential for the processing of rare repeat transcripts that escape transcriptional silencing. Indeed, they found that a point mutation in the H3-Lys9-binding domain of Chp1, or deletion of the Clr4 methyltransferase, not only prevented RITS from binding to chromatin but also abolished RITS-associated siRNAs. Moreover, loss of Clr4 caused centromeric-repeat transcripts to accumulate. So RITS needs to be associated with chromatin to process transcripts into siRNAs and to generate heterochromatin.

The model proposed by the authors establishes a mechanism through which RITS can function in cis to bring about effective gene silencing. Heterochromatic marks are initially established in a Dcr-dependent manner, and therefore presumably through the RNAi pathway. Such siRNAs somehow guide Clr4 and factors that mediate heterochromatin assembly to homologous target sequences. RITS then localizes at these specific loci and mediates the processing of nascent RNA transcripts, probably by recruiting Dcr or RNA-dependent RNA polymerase. This thereby generates more siRNAs — so the mechanism continues...